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176. Development of an siRNA-Based Therapeutic for Hypercholesterolemia
METABOLIC DISEASES I 174. The Importance of Transgene Optimization in Correcting the Dyslipidemia in a Mouse Model of Familial Hypercholesterolemia by Gene Therapy with AAV8
Sadik H. Kassim,1 Luk H. Vandenberghe,1 Ru Ziao,1 Dawn Marchadier,2 Peter Bell, Debra Cromley,2 James M. Wilson, Daniel J. Rader.2 1 Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA. Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. FH patients are refractory to traditional pharmacologic therapy; a continuous regimen of LDL apheresis is the most effective therapy to date. The success of orthotopic liver transplantation in correcting the metabolic abnormalities of homozygous FH patients suggests the liver to be an appropriate target for somatic gene transfer in the treatment of this disease. Inbred mice with germ line interruptions in the LDLR and apobec-1 genes (LDLR//Apobec-/-) develop severe hypercholesterolemia and extensive atherosclerosis on a chow diet. These mice effectively simulate the metabolic and clinical aspects of FH and serve as excellent animal models for the testing of gene therapeutic approaches. AAV8 vectors encoding mouse LDLR (mLDLR) or human LDLR (hLDLR) or nuclear beta galactosidase (nLacZ) under the control of a liver-specific human thyroid binding globulin gene promoter (TBG) were used for intravenous (I.V.) gene transfer into the liver of (LDLR-/-/ApoB-/-) mice. Mice were I.V. injected with a range of doses from 1 1012 genome copies (GCs) to 1 1010 GCs of vector per mouse. TBG-hLDLR and TBG-nLacZ treated mice demonstrated no significant reductions in serum triglyceride or cholesterol levels at all time points tested. By contrast, TBG-mLDLR-treated mice demonstrated an average of 79 ± 8% decrease in serum cholesterol levels as early as one week after treatment. AAV8.TBG.mLDLR was effective at doses as low as 1 1010 GCs of vector per mouse. These results were durable and extended to past 100 days post-treatment. Clinical chemistry and histological analyses will also be discussed. There was no evidence of immune activation against the vector or transgene in any group. Collectively, these data demonstrate the safety and potential benefits of the liver-specific AAV8 vector in the treatment of familial hypercholesterolemia using in vivo liver gene therapy and show how important it is to optimize transgene and/or use a transgene that is syngeneic to the recipient model.
175. Rapid Correction of Ornithine Transcarbamylase Deficiency (OTCD) in OTCD Mouse Model by Self-Complementary AAV2/8 Vector
Huan Wang,1,4 Lili Wang,1 Hiroki Morizono,2 David Jones,2 Peter L. Bell,1 A. Avilan Dorante,1 Guangping Gao,3 Mark L. Batshaw,2 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Center for Genetic Medicine Research, Children’s National Medical Center, Children’s Research Institute, Washington, DC; 3Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 4Vaccine Research Institute, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. Ornithine transcarbamylase is an enzyme involved in the catabolism of nitrogen into urea. A deficiency of this X-linked inborn error of metabolism is associated with protein intolerance and episodes of life Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
threatening hyperammonemia. A severe deficiency of OTC in males manifests with hyperammonemic coma in the neonatal period and, if untreated, is rapidly fatal. Even with current therapy involving dialysis and alternate pathway therapy, there is almost a 50% mortality rate in neonates. In our previous experiment, we demonstrated that in the OTCD sparse fur mouse (spf), AAV7, 8, and 9 vectors expressing the mouse OTC gene were able to restore urinary orotate levels (a biomarker for OTC activity) to normal 15 days after intraportal vector infusion. Efficacy persisted 1 year post-treatment. Treated mice had substantially higher OTC enzyme activity in liver and were able to tolerate an ammonia challenge that, for untreated mice, was lethal. However, these therapeutic effects were achieved only at a high vector dose of 1x1012 genome copy (GC)/mouse, which is equivalent to 4x1013 GC/kg for treatment of humans. In addition, a more rapid therapeutic effect is desirable for this life-threatening disease that presents in the first week of life. Self-complementary vectors could circumvent a rate-limiting step: the conversion of the single stranded vector DNA to double stranded DNA. We hypothesize that this would not only increase the gene transfer efficiency but also improve expression kinetics. We constructed AAV2/8sc.TBG.mOTC, a selfcomplementary AAV vector containing the mouse OTC gene driven by a liver-specific TBG promoter and packaged with AAV8 capsid. Three days after a single intravenous injection at the dose of 1x1011 GC or 3x1011 GC, partial reduction of urine orotate was observed in OTCD (spf-ash) mice in both dose groups. Complete normalization of urine orotate was achieved 7 days after injection. This suggests AAV2/8sc.TBG.mOTC is a promising vector for gene therapy of OTCD with high efficiency and fast kinetics.
176. Development of an siRNA-Based Therapeutic for Hypercholesterolemia
Amy C. H. Lee,1 Sean Semple,1 Merete Eisenhardt,1 Jodi Ell,1 Lloyd Jeffs,1 David Le,1 Matthew Lee,1 Josephine Leung,1 Kevin McClintock,1 Janet Phelps,1 Stephen Reid,1 Marjorie Robbins,1 Tracey Sutcliffe,1 Lisa Wood,1 Ed Yaworski,1 Ian MacLachlan.1 1 Tekmira Pharmaceuticals Corporation, Burnaby, BC, Canada. The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of novel therapeutics against targets previously thought to be “nondruggable” with conventional medicines. Hurdles to achieving this therapeutic potential include the delivery of small interfering RNAs (siRNAs) to target tissue in a clinically relevant manner. Here we show that siRNAs, when delivered systemically in a Stable Nucleic Acid Lipid Particle (SNALP), potently silence the conventionally non-druggable disease target, apolipoprotein B (apoB), in rodents and non-human primates. ApoB-specific siRNAs were encapsulated in SNALP and administered by intravenous injection. A single siRNA injection resulted in durable (more than one month), dose-dependent silencing of apoB mRNA expression in the liver. ApoB silencing was demonstrated to occur through RISC mediated cleavage of the apoB mRNA at precisely the site predicted for the RNAi mechanism and duration of effect correlated with preferential maintenance of the antisense strand of the siRNA duplex, as measured by stem-loop RTqPCR. Improvements in SNALP formulation technology resulted in an increase in the therapeutic index such that significant reductions in apoB expression were achieved at doses more than one thousand fold lower than those that elicit toxicity in preclinical models. Significant RNAi mediated reductions in apoB protein, serum cholesterol, and low-density lipoprotein (LDL) levels were found to protect LDL receptor knockout mice from developing atherosclerotic lesions when fed a high fat, so-called ‘Western’ type diet. These findings demonstrate clinically relevant RNAi-mediated gene silencing and support the potential of RNAi therapeutics as a new class of drugs.
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Sadik H. Kassim,1 Luk H. Vandenberghe,1 Ru Ziao,1 Dawn Marchadier,2 Peter Bell, Debra Cromley,2 James M. Wilson, Daniel J. Rader.2 1 Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA. Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. FH patients are refractory to traditional pharmacologic therapy; a continuous regimen of LDL apheresis is the most effective therapy to date. The success of orthotopic liver transplantation in correcting the metabolic abnormalities of homozygous FH patients suggests the liver to be an appropriate target for somatic gene transfer in the treatment of this disease. Inbred mice with germ line interruptions in the LDLR and apobec-1 genes (LDLR//Apobec-/-) develop severe hypercholesterolemia and extensive atherosclerosis on a chow diet. These mice effectively simulate the metabolic and clinical aspects of FH and serve as excellent animal models for the testing of gene therapeutic approaches. AAV8 vectors encoding mouse LDLR (mLDLR) or human LDLR (hLDLR) or nuclear beta galactosidase (nLacZ) under the control of a liver-specific human thyroid binding globulin gene promoter (TBG) were used for intravenous (I.V.) gene transfer into the liver of (LDLR-/-/ApoB-/-) mice. Mice were I.V. injected with a range of doses from 1 1012 genome copies (GCs) to 1 1010 GCs of vector per mouse. TBG-hLDLR and TBG-nLacZ treated mice demonstrated no significant reductions in serum triglyceride or cholesterol levels at all time points tested. By contrast, TBG-mLDLR-treated mice demonstrated an average of 79 ± 8% decrease in serum cholesterol levels as early as one week after treatment. AAV8.TBG.mLDLR was effective at doses as low as 1 1010 GCs of vector per mouse. These results were durable and extended to past 100 days post-treatment. Clinical chemistry and histological analyses will also be discussed. There was no evidence of immune activation against the vector or transgene in any group. Collectively, these data demonstrate the safety and potential benefits of the liver-specific AAV8 vector in the treatment of familial hypercholesterolemia using in vivo liver gene therapy and show how important it is to optimize transgene and/or use a transgene that is syngeneic to the recipient model.
175. Rapid Correction of Ornithine Transcarbamylase Deficiency (OTCD) in OTCD Mouse Model by Self-Complementary AAV2/8 Vector
Huan Wang,1,4 Lili Wang,1 Hiroki Morizono,2 David Jones,2 Peter L. Bell,1 A. Avilan Dorante,1 Guangping Gao,3 Mark L. Batshaw,2 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Center for Genetic Medicine Research, Children’s National Medical Center, Children’s Research Institute, Washington, DC; 3Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 4Vaccine Research Institute, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. Ornithine transcarbamylase is an enzyme involved in the catabolism of nitrogen into urea. A deficiency of this X-linked inborn error of metabolism is associated with protein intolerance and episodes of life Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
threatening hyperammonemia. A severe deficiency of OTC in males manifests with hyperammonemic coma in the neonatal period and, if untreated, is rapidly fatal. Even with current therapy involving dialysis and alternate pathway therapy, there is almost a 50% mortality rate in neonates. In our previous experiment, we demonstrated that in the OTCD sparse fur mouse (spf), AAV7, 8, and 9 vectors expressing the mouse OTC gene were able to restore urinary orotate levels (a biomarker for OTC activity) to normal 15 days after intraportal vector infusion. Efficacy persisted 1 year post-treatment. Treated mice had substantially higher OTC enzyme activity in liver and were able to tolerate an ammonia challenge that, for untreated mice, was lethal. However, these therapeutic effects were achieved only at a high vector dose of 1x1012 genome copy (GC)/mouse, which is equivalent to 4x1013 GC/kg for treatment of humans. In addition, a more rapid therapeutic effect is desirable for this life-threatening disease that presents in the first week of life. Self-complementary vectors could circumvent a rate-limiting step: the conversion of the single stranded vector DNA to double stranded DNA. We hypothesize that this would not only increase the gene transfer efficiency but also improve expression kinetics. We constructed AAV2/8sc.TBG.mOTC, a selfcomplementary AAV vector containing the mouse OTC gene driven by a liver-specific TBG promoter and packaged with AAV8 capsid. Three days after a single intravenous injection at the dose of 1x1011 GC or 3x1011 GC, partial reduction of urine orotate was observed in OTCD (spf-ash) mice in both dose groups. Complete normalization of urine orotate was achieved 7 days after injection. This suggests AAV2/8sc.TBG.mOTC is a promising vector for gene therapy of OTCD with high efficiency and fast kinetics.
176. Development of an siRNA-Based Therapeutic for Hypercholesterolemia
Amy C. H. Lee,1 Sean Semple,1 Merete Eisenhardt,1 Jodi Ell,1 Lloyd Jeffs,1 David Le,1 Matthew Lee,1 Josephine Leung,1 Kevin McClintock,1 Janet Phelps,1 Stephen Reid,1 Marjorie Robbins,1 Tracey Sutcliffe,1 Lisa Wood,1 Ed Yaworski,1 Ian MacLachlan.1 1 Tekmira Pharmaceuticals Corporation, Burnaby, BC, Canada. The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of novel therapeutics against targets previously thought to be “nondruggable” with conventional medicines. Hurdles to achieving this therapeutic potential include the delivery of small interfering RNAs (siRNAs) to target tissue in a clinically relevant manner. Here we show that siRNAs, when delivered systemically in a Stable Nucleic Acid Lipid Particle (SNALP), potently silence the conventionally non-druggable disease target, apolipoprotein B (apoB), in rodents and non-human primates. ApoB-specific siRNAs were encapsulated in SNALP and administered by intravenous injection. A single siRNA injection resulted in durable (more than one month), dose-dependent silencing of apoB mRNA expression in the liver. ApoB silencing was demonstrated to occur through RISC mediated cleavage of the apoB mRNA at precisely the site predicted for the RNAi mechanism and duration of effect correlated with preferential maintenance of the antisense strand of the siRNA duplex, as measured by stem-loop RTqPCR. Improvements in SNALP formulation technology resulted in an increase in the therapeutic index such that significant reductions in apoB expression were achieved at doses more than one thousand fold lower than those that elicit toxicity in preclinical models. Significant RNAi mediated reductions in apoB protein, serum cholesterol, and low-density lipoprotein (LDL) levels were found to protect LDL receptor knockout mice from developing atherosclerotic lesions when fed a high fat, so-called ‘Western’ type diet. These findings demonstrate clinically relevant RNAi-mediated gene silencing and support the potential of RNAi therapeutics as a new class of drugs.
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